The goal of this project is to identify molecular mechanisms involved in the regulation of the beta-adrenergic receptor (bAR)-coupled adenylylcyclase. Although there are three bAR subtypes, b1AR, b2AR and b3AR, the physiological relevance of each remains to be elucidated. One possibility is that they may be regulated differently by agonists and other modulators. At least three major mechanisms of receptor regulation have been identified: desensitization, sequestration, and down-regulation. We and others have shown that for the three human subtypes, the ability to undergo regulation is b2AR >b1AR>> b3AR. To further explore the differences between human b1AR and b2AR, we modified their cDNAs to contain an N-terminal epitope (the FLAG epitope, FL) and inserted them (b1ARFL and b2ARFL) into the expression vector ZEM228c. Using the latter, we stably expressed FL-tagged bAR in transfected Chinese hamster fibroblasts (CHW-hb1FL and -hb2FL cells). As receptor expression is under control of the metallothionein promotor, we can manipulate cellular bAR levels by varying the zinc sulfate concentration in the culture medium. We first established that each bARFL subtype functioned the same as wild-type bAR by demonstrating that it coupled to adenylyl cyclase and underwent desensitization, sequestration, and down-regulation. When membranes from CHW-hb1FL and -hb2FL cells were analyzed by Western blotting with anti-FL antibodies (Ab), a single major protein was detected in each with essentially the same mobility as the wild-type bAR. We are using anti-FL Ab to immunoaffinity purify bARFL from control and desensitized cells. It is well established that agonist-mediated desensitization involves phosphorylation of bAR by protein kinase A (PKA) and bAR kinase. bAR also can be phosphorylated and desensitized by protein kinase C (PKC). To correlate phosphorylation with desensitization, we are taking a novel approach and using Western blotting with anti-phosphoamino acid Ab to detect phosphorylation of the receptors during desensitization. This has the great advantage of not having to label the cells with large amounts of 32-P which has been the traditional approach. Although our initially results are encouraging, immunostaining of the receptors (so far b2ARFL) with antiphosphoserine was weak relative to staining with anti-FL. We are also using the CHW-hb2FL cells to explore the fate of the receptor protein during down-regulation. When the latter cells were exposed to agonist, binding activity in cell lysates decreased with time and was similar to that observed in CHW-hb2 cells. Exposing the cells to a permeable cAMP derivative also led to down-regulation but it was slower and less extensive. Cyclic AMP-mediated down-regulation appears to be due to destabilization of the b2AR mRNA and to be mediated by a cAMP-induced mRNA binding protein. Thus, in cells exposed to agonist, there are two components of down-regulation: a cAMP-dependent reduction in bAR mRNA and subsequently bAR synthesis; and an agonist-dependent loss of bAR binding. We are interested in the latter process which may be due either to bAR degradation or to inactivation. Down-regulation was further complicated by the level of receptor expression. Cells expressing b2AR high levels exhibited less down-regulation than cells expressing low b2AR levels. A second aspect of bAR down-regulation involves receptor mRNA levels. We had shown previously that b1AR mRNA undergoes down-regulation in rat C6 glioma cells exposed to either agonist or agents that increase cAMP levels. We established that this down-regulation is due to repression of gene transcription and is mediated by the inducible cAMP early repressor (ICER), a member of the cAMP response element modulatory protein (CREM) family of transcription factors. Now we have found that exposure of C6 cells to phorbol esters also caused a down-regulation of b1AR mRNA. Down- regulation was due to activation of PKC as it was blocked in cells pre- treated with a specific PKC inhibitor. As the half-life of b1AR mRNA was similar in control and phorbol ester-treated cells, down-regulation was not due to mRNA destabilization. Using rat b1AR gene promotor/luciferase reporter constructs, we established that down-regulation of b1AR mRNA was due to repression of gene transcription and identified the promotor sequence as TGACGCGC (-343 to -336 of the 5' flanking region). Although this sequence, designated as a PRE (phorbol ester response element), has partial homology to the CRE found in the rat b1AR promotor, mutation of the former but not the latter prevented the phorbol ester-mediated reduction in reporter activity. Using gel mobility shift assays, we found C6 cell extracts formed two major complexes with a labeled DNA oligonucleotide containing the b l AR PRE sequence. As similar levels were found in control and phorbol ester-treated cells, the DNA binding proteins are not induced by the phorbol ester. This was consistent with the observation that phorbol ester-mediated down-regulation of b1AR mRNA in C6 cells was not blocked by inhibition of protein synthesis. Our working hypothesis is that PKC-mediated phosphorylation activates one or more transcription factors that bind to the PRE sequence in the rat b1AR promotor and repress transcription. We have found a highly homologous sequence in a similar location of the human b1AR promotor. Finally, we are exploring the down-regulation of human b1AR and b1AR mRNA in stably transfected CHW cells. To quantify b1AR mRNA levels, we are using a non-radioactive ribonuclease protection assay. Briefly, total RNA is extracted from the cells, hybridized with an excess of a biotinylated riboprobe (an antisense sequence synthesized from the b1AR cDNA), and digested with ribonucleases. The resistant hybrids are separated by gel electrophoresis, transferred to nylon membranes and detected with strepavidin-conjugated alkaline phosphatase and chemiluminescence. As was the case for b2AR in CHW cells, we found that down-regulation was mediated to a greater extent by agonist than by permeable cAMP derivatives, and was inversely dependent on receptor levels. Using ribonuclease protection assays, we observed b1AR mRNA levels decreased in both low and high expressing cells. As the cells were transfected with a cDNA lacking endogenous promoter sequences, down-regulation of receptor mRNA is mostly likely due to cAMP-mediated destabilization as reported for human and hamster b2AR. In contrast, agonist-mediated down-regulation appears to involve the receptor itself and to be dependent on receptor levels.